Measuring peptide concentration

Artem Evdokimov AEVDOKIMOZ at cinci.rr.com
Wed Mar 20 21:36:25 EST 2002


Hi,

Cystine has significant absorption at 280 whereas free cysteine does not :)

As a general rule, one S-S bond in otherwise absorption-free peptide will
give you something like 130 M-1 cm-1 in extinction coefficient. Unpaired
cysteine gives nearly zero. For comparison, a single tyrosine or tryptophane
give a whopping 1300 and 6000 respectively !

For your hypothetical (average m.w., assuming each aa is about 110 Da)
25-mer, with two Cys, this will result in approximately 0.05 OD for 1 mg/ml,
1cm path. Without the disulphide, A280 should be nearly zero. In comparison,
a single Trp gives 2.7 OD for 1 mg/ml, and a single Tyr gives 0.65. (All
values approximate, of course!).

In conclusion, I won't trust this method as a reliable tool for SH
determination - I'd rather use Ellman's reagent :)

Artem





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